Although the use of intensified treatment regimens has improved outcomes for patients with typical T-cell acute lymphoblastic leukemia (T-ALL), outcomes remain dismal for patients whose leukemic blasts express markers of differentiation arrest at the earliest stages of T-cell development. The mechanisms responsible for chemotherapy resistance in these cases are unknown. Conventional combination chemotherapy is designed to induce diverse death-promoting signals that converge on the mitochondrial apoptotic machinery, and we have now shown that pre-treatment resistance to mitochondrial apoptosis is a cellular phenotype that predicts treatment failure in T-ALL. However, the molecular mechanisms underlying this apoptosis-resistant cellular phenotype are poorly defined, and this knowledge gap represents the primary obstacle to development of a rational therapeutic strategy to reverse apoptosis resistance and restore chemosensitivity in high-risk T-ALL. We have now shown that apoptosis resistance closely correlates with inactivating mutations of EZH2, a core component of the polycomb repressive complex 2 transcriptional complex. Moreover, T-ALL cases with differentiation arrest at very early stages of T-cell development are selectively sensitive to BCL2 inhibition, a dependence that reflects the cellular context of the non-malignant counterpart of these cells. Our overarching premise is that the most significant improvements in outcome for patients with T-ALL will be achieved by defining the molecular mechanisms responsible for resistance to conventional chemotherapy, and leveraging this knowledge to develop therapeutic interventions to restore chemosensitivity. We propose to achieve this through investigation of the following Specific Aims: 1) Define the mechanisms through which EZH2 inactivation induces resistance to mitochondrial apoptosis, and 2) Develop an effective therapeutic strategy to reverse apoptosis resistance and restore chemosensitivity in high-risk T-ALL. The significance of the proposed research is that it is expected to provide an answer to what is, in our opinion the major unanswered question in clinical oncology: why do different patients with seemingly identical tumors exhibit strikingly differences clinical responses to conventional chemotherapy, with some patients achieving long-term cures, and others suffering failure to achieve even a temporary remission? Successful completion of this proposal is expected to provide an answer to this question, and lead to a clinical trial of a rationally designed therapeutic strategy to restore chemosensitivity in this particularly aggressive subtype of leukemia. More broadly, this approach will provide a paradigm for the rational development of innovative clinical trials to reverse chemoresistance and improve clinical outcomes for patients with chemoresistant tumors.